Oleaginous yeast are defined as those organisms that are naturally capable of oil synthesis and accumulation, wherein oil accumulation ranges from at least about 25% up to about 80% of the cellular dry weight. Genera typically identified as oleaginous yeast include, but are not limited to: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces. More specifically, illustrative oil-synthesizing yeast include: Rhodosporidium toruloides, Lipomyces starkeyii, L. lipoferus, Candida revkaufi, C. pulcherrima, C. tropicalis, C. utilis, Trichosporon pullans, T. cutaneum, Rhodotorula glutinus, R. graminis and Yarrowia lipolytica (formerly classified as Candida lipolytica).
The technology for growing oleaginous yeast with high oil content is well developed (for example, see EP 0 005 277B1; Ratledge, C., Prog. Ind. Microbiol. 16:119-206 (1982)). And, these organisms have been commercially used for a variety of purposes in the past. For example, various strains of Yarrowa lipolytica have historically been used for the manufacture and production of: isocitrate lyase; lipases; polyhydroxy-alkanoates; citric acid; erythritol; 2-oxoglutaric acid; γ-decalactone; γ-dodecalactone; and pyruvic acid. More recently, however, the natural abilities of oleaginous yeast have been enhanced by advances in genetic engineering, resulting in organisms capable of producing polyunsaturated fatty acids (“PUFAs”). Specifically, Picataggio et al. have demonstrated that Y. lipolytica can be engineered for production of ω-3 and ω-6 fatty acids, by introducing and expressing genes encoding the ω-3/ω-6 biosynthetic pathway (see WO 2004/101757).
Recombinant production of any heterologous protein is generally accomplished by constructing an expression cassette in which the DNA coding for the protein of interest is placed under the control of appropriate regulatory sequences (i.e., promoters) suitable for the host cell. The expression cassette is then introduced into the host cell (usually by plasmid-mediated transformation or targeted integration into the host genome) and production of the heterologous protein is achieved by culturing the transformed host cell under conditions necessary for the proper function of the promoter contained within the expression cassette. Thus, the development of new host cells (e.g., oleaginous yeast) for recombinant production of proteins generally requires the availability of promoters that are suitable for controlling the expression of a protein of interest in the host cell.                A variety of strong promoters have been isolated from Yarrowia lipolytica that are useful for heterologous gene expression in yeast. For example, U.S. Pat. No. 4,937,189 and EP220864 (Davidow et al.) disclose the sequence of the XPR2 gene (which encodes an inducible alkaline extracellular protease) and upstream promoter region for use in expression of heterologous proteins. U.S. Pat. No. 6,265,185 (Muller et al.) describe promoters for the translation elongation factor EF1-α (TEF) protein and ribosomal protein S7 that are suitable for expression cloning in yeast and heterologous expression of proteins. These promoters were improved relative to the XPR2 promoter, when tested for yeast promoter activity on growth plates (Example 9, U.S. Pat. No. 6,265,185) and based on their activity in the pH range of 4-11. WO 2005/003310 and commonly owned co-pending U.S. patent application Ser. No. 11/183664 describe regulatory sequences (e.g., promoters, introns) of the glyceraldehyde-3-phosphate dehydrogenase (gpd) and phosphoglycerate mutase (gpm) genes; and, WO 2005/049805 describes regulatory sequences (e.g., promoters, introns) of the fructose-bisphosphate aldolase (fba) gene. Similarly, Juretzek et al. (Biotech. Bioprocess Eng., 5:320-326 (2000)) compares the glycerol-3-phosphate dehydrogenase (G3P), isocitrate lyase (ICL1), 3-oxo-acyl-CoA thiolase (POT1) and acyl-CoA oxidase (POX1, POX2 and POX5) promoters with respect to their regulation and activities during growth on different carbon sources.        
Despite the utility of these known promoters, however, there is a need for new improved yeast promoters for metabolic engineering of yeast (oleaginous and non-oleaginous) and for controlling the expression of heterologous genes in yeast. Furthermore, possession of a suite of promoters that are regulatable under a variety of natural growth and induction conditions in yeast will play an important role in industrial settings, wherein it is desirable to express heterologous polypeptides in commercial quantities in said hosts for economical production of those polypeptides. Thus, it is an object of the present invention to provide such promoters that will be useful for gene expression in a variety of yeast cultures, and preferably in Yarrowia sp. cultures and other oleaginous yeast.
Applicants have solved the stated problem by identifying the gene (gpat) encoding glycerol-3-phosphate O-acyltransferase (GPAT) from Yarrowia lipolytica and the promoter responsible for driving expression of this native gene. The promoter is useful for expression of heterologous genes in Yarrowia and has improved activity with respect to the TEF promoter.